1. Field of Invention
The present invention relates to stable glutamine derivatives and their use in rehydration and nutrition therapy and for enhanced nutrition in animals. More particularly the present invention is directed to the bioproduction of polypeptides comprising glutamine or glutamine rich regions and use of these polypeptides for rehydration and nutrition therapy and enhanced nutrition in animals.
2. Background of Invention
Glutamine is an amino acid which cotransports Na+ across the enterocyte brush border membrane. It is known to be the major bowel nutrient and energy source and has been used in intravenous solutions to improve nitrogen balance, inhibit protein breakdown, stimulate the growth of epithelial cells, and reduce intestinal villous atrophy. In addition, various researchers have shown that glutamine stimulates the absorption of sodium and chloride and have used glutamine in oral rehydration solutions to reduce cholera diarrhea.
Viral enteritis is a leading cause of diarrhea in infants and toddlers less than two years old. Each year in the United States, about 22,000 infants are hospitalized for treatment of rotavirus-induced dehydration. In a majority of the cases, diarrheal disease morbidity and mortality is due to dehydration. The primary effect seen is the loss of fluid and electrolytes in diarrheal stools. An immediate effect in treatment of dehydration can be achieved by early oral administration of sugar (glucose) and electrolyte solution and continued feeding. However, conventional therapy by administration of oral rehydration formulations does not reduce stool volume or the duration of diarrhea. Thus, modifications of the oral rehydration therapy are needed to actually reduce stool volumes or speed the recovery of normal mucosal function, which in term will substantially enhance the acceptability and effectiveness of such therapy. The effects of organic compounds of salt and water absorption were first applied successfully to the treatment of patients with chloera and thereafter it was shown experimentally that the salt substrate cotransport was substantially intact in cholera patients and that oral therapy with sodium, chloride, potassium, biocarbonate and glucose in the same solution will restore and maintain normal blood volume and electrolyte concentrations, organic molecules such as D-hexoses, neutral amino acids, dipeptides and tripeptides of neutral amino acids, and water soluble vitamins can also enhance sodium absorption, following by water absorption in the small intestines. The present inventors have previously shown the efficacy of glutamine in intestinal sodium absorption. (Lima et al., Brazilian J. Med. Biol Res., 25; 637-640, (1992)). However, the greatest limitations to the oral use of glutamine is its instability and tendency to degrade in water and acid, conditions which are found in the stomach.
Bone marrow transplantation is being increasingly used in the treatment of hematologic malignancies. Patients undergoing bone marrow transplantation loose body protein because of the catabolic of acts of chemotherapy, total body radiation and graft-versus host disease. In addition, gastrointestinal toxicity often limits the consumption absorption of enteral nutrients. Infectious complications also remain a major cause of morbidity of these patients. Infections accelerate protein loss, and protein-calories malnutrition may decrease host resistance to microbial invasion. Parenteral nutrition is known to attenuate such protein losses and may prevent complications associated with malnutrition. Despite use in many centers, parenteral nutrition is also, unfortunately, associated with an increased incidence of infection in patients receiving chemotherapy with or without the irradiation, and also in those receiving allogeneic bone marrow transplantation. Further, despite conventional nutritional support, these patients still suffer from markedly negative nitrogen balance.
Modification of amino acid formulation may improve the clinical and metabolic efficacy of parentenal nutrition. Notably absent in all commercially available parenteral nutrient solutions is glutamine, because it has a shorter shelf-life than the commonly used amino acids and has been considered a non-essential amino acid. However, during catabolic states, glutamine concentrations in intracelluar pools (primarily skeletal muscle) fall rapidly. This reduction in glutamine occurs due to use of glutamine for renal ammoniagenis and as oxidizable fuel for stimulated lymphocytes and macrophages and intestinal muscosal cells. Glutamine-enriched parenteral or enteral nutrition has been shown to enhance nitrogen balance, attenuate intestinal mucosal damage, decrease bactermia and improve survival after radiation and chemotherapy when compared with glutamine-free nutrition. Limited clinical studies in postoperative patients have shown improved nitrogen retention with glutamine-enriched parenteral feeding. The clinical safety of L-glutamine added as a component of balanced parenteral nutrient solutions has recently been documented (C. Ziegler et al., Annals of Internal Medicine, 116:821-828 (1992) and references cited therein incorporated herein by reference in its entirety).
In addition to the therapeutic value of L-glutamine in rehydration therapy and nutrition of diseased individuals, glutamine also has potential for a nutritional enhancement in healthy individuals. A stable glutamine derivative may form the basis for anabolic nutrient formulations for the building and sustaining of muscle mass in humans or other animals. For example these formulations may be desirable for use in connection with body building and athletic activities in humans and may be useful in optimizing feed rations for livestock.
While the efficacy of glutamine in rehydration and nutrition therapies and for nutritional enhancements is known, the instability of glutamine in the digestive tract has diminished its usefulness. Accordingly, there is a need for a method for administration of glutamine to humans or other animals which will provide effective treatment in oral rehydration and nutrition therapy or nutritional enhancements, while overcoming the difficulties of instability in acidic environment.
It has been previously reported that glutamine (GLN) and alanyl-glutamine (ALA-GLN) can be chemically synthesized and used to treat conditions associated with dehydration or nitrogen deficiency-based malnutrition. (See U.S. Pat. No. 5,561,111 to Guerrant et al. directed to Stable Glutamine Derivatives for Oral and Intravenous Rehydration and Therapy (ORNT) and U.S. patent application Ser. No. 09/527329 incorporated herein by reference in their entirety.) However, a biological production method could offer the advantages over chemical production methods in the preparation of glutamine rich peptides, oligopeptides and proteins for use in rehydration, and nutrition therapy and nutritional enhancement.
Accordingly, the present invention is directed to providing new stable glutamine derivatives capable of delivering glutamine to the body in oral or intravenous rehydration and nutrition therapy or in nutritional enhancement. In accordance with one embodiment of the invention the glutamine derivatives are expressed in a bacteria, yeast, plant or animal host cell that is part of a larger polypeptide. The proteins can be expressed under the control of a constitutive promoter or an inducible or developmentally regulated promoter. The expressed modified polypeptide can then be administered in either a purified form, an enriched formulation or in a non-purified form (i.e. administered in the cells in which the polypeptide was expressed) as an improved method for treating conditions associated with dehydration or nitrogen deficiency based malnutrition or as a nutritional supplement.
An objective of the present invention is a method for the bioengineered production of glutamine rich peptides which contain a principal component of glutamine (for example (GLN)n and ALA(GLN)n) in one embodiment or in an alternative embodiment alanyl-glutamine (ALA-GLN)n. These peptides may exist as peptides or be coupled with other like or different units to form oligopeptides, and/or proteins in prokaryotic or eukaryotic cells.
The method provides for the production of biologically relevant quantities of peptides, oligopeptides, and proteins containing one or more amino acid coupled with glutamine or polyglutamines tracts, with and without internal protease cleavage sites, by overexpression of DNA sequences encoding these peptides and the like in prokaryotic and eukaryotic cells including but not limited to bacteria (e.g., Escherichia coli, Pseudomonas sps., etc.), photosynthetic bacteria (e.g., Synechocystis sps. etc.), algae (e.g., Chlamydomonas, Chlorella, etc.), yeast (e.g., Saccharomyces sps.), plants (Nicoliana tabacum, Zea mays, Glycine max, etc.) and animal cells (e.g., BHK, 3T3) either cultured or as part of transgenic organisms, under the control of a constitutive promoter (i.e., gene transcription regulatory sequence(s)), an inducible or a developmentally-regulated promoter.
Additionally, the method provides for the bioengineered production of such GLN, ALA-GLN and GLN-rich peptides for example (GLN)n, ALA(GLN)n and (ALA-GLN)n, oligopeptides, and/or protein and the like, coupled to other existing cellular peptides or proteins which stabilize or enhance their formation or accumulation in the cell. The bioengineered peptides may be fused to another protein either at the C- or N-terminus. For example, this includes fusion to sequences for intracellular/intercellular localization (targeting sequences) and secretory sequences.
A further objective of the invention is to produce compositions of materials that result from the bioengineering process including nucleic acid molecules which code for at least one glutamine rich peptide, glutamine rich polypeptides produced by the bioengineering method, glutamine rich oligopeptides and glutamine rich proteins derivatives of glutamine rich polypeptides produced by the bioengineering method and the genetically modified strains of bacteria (e.g., Escherichia coli, Pseudomonas sps., etc.), photosynthetic bacteria (e.g., Synechocystis sps. etc.), algae (e.g., Chlamydononas, Chlorella, etc.), yeast (e.g., Saccharomyces sps.), plants (Nicotiana tabacum, Zea mays, Glycine max, etc.) and animals (e.g., BHK, 3T3), either cultured or as part of transgenic organisms, that express the proteins.
A further objective of the invention is a method for rehydration therapy comprising administering to a patent in need thereof an effective amount of biologically produced at least one glutamine rich peptide for example a peptide comprising the sequence of (GLN)n, (ALA-GLN)n or ALA(GLN)n. The biologically produced peptide may be a pure peptide, part of an oligopeptide comprised of like peptides, unlike peptides or a mixture thereof, or part of a protein comprised of like peptides, unlike peptides or a mixture thereof and may be administered in a purified form, and enriched formulation, or a non-purified form (i.e. administered in the cells or tissues in which the peptides, oligopeptides, or polypeptides are expressed).
A further objective of the invention is a method for nutrition therapy comprising administering to a patent in need thereof an effective amount of biologically produced at least one glutamine rich peptide for example a peptide comprising the sequence of (GLN)n, (ALA-GLN)n or ALA(GLN)n. The biologically produced peptide may be a pure peptide, part of an oligopeptide comprised of like peptides, unlike peptides or a mixture thereof, or part of a protein comprised of like peptides, unlike peptides or a mixture thereof and may be administered in a purified form, and enriched formulation or a non-purified form (e.g., administered in the cells or tissues in which the peptides, oligopeptides, or polypeptides are expressed).
A further objective of the invention is a method for building muscle mass in a healthy mammal comprising administering to an individual a biologically produced at least one glutamine rich peptide comprising for example a peptide comprising the sequence of (GLN)n, (ALA-GLN)n or ALA(GLN)n. The biologically produced peptide may be a pure peptide, part of an oligopeptide comprised of like peptides, unlike peptides or a mixture thereof, or part of a protein comprised of like peptides, unlike peptides or a mixture thereof and may be administered in a purified form, and enriched formulation or a non-purified form (i e. administered in the cells or tissues in which the peptides, oligopeptides, or polypeptides are expressed).